Biochip is a term used to refer to an array of discrete domains containing biochemical probes that will react with desired targets or analytes. Some means is generally necessary to quantify the amount of bound target, and it is common to employ some form of photon detection. Fluorescence or chemo-luminescence can be employed with spatially resolved fluorescence scanning being by far the most common detection approach. Such a fluorescence imaging system is described in detail in U.S. Pat. No. 5,672,880 (Sep. 30, 1997).
Because of the very small quantities of analyte normally encountered, highly sensitive means are required to detect photon emissions. A large industry has developed around producing scanners used in the detection of signals from biochips.
The present invention is based on the concept that constructive interference of an emission or excitation signal can occur with thin (e.g. ¼-wavelength) dielectrics on top of a reflective surface, such as aluminum or other metal. Constructive interference permits enhanced amplification of the signal over and above what may be achieved without a reflector, or beyond that which may hold for an incoherent radiation model. To create constructive interference, the fluorophore must lie at a particular distance from the surface of the mirror.
Kain, et al. in U.S. Pat. Nos. 6,008,892 and 6,177,990 disclose an approach to enhancing the detection of fluorescence from targets that are flat with respect to a wavelength. Their approach uses reflective substrates with a dielectric coating that is an odd multiple of a ¼ wavelength. More specifically, they teach the concept of creating constructive interference of the excitation, by employing a dielectric thickness that is ¼+N/2 wavelengths, where N is an integer greater than or equal to 0. They note that their approach yields substantial amplification of emission signal, with resultant enhancement of signal to noise ratio (SNR). The majority of biochips in current use tend to yield analyte distribution that is relatively flat with respect to an emission wavelength, making their invention effective in these cases. The aluminum surface eliminates background autofluorescence of underlying substrate. The approach amplifies background fluorescence from contaminants that are coplanar with the analyte regions. This form of background, often caused by failure to remove traces of excess flurophore, is amplified by the same factor as the desired signal.
Because a major problem with biochips is the presence of spurious background signal, much of which arises from the plane containing the standard analyte domains, i.e. the plane of attachment, the use of such a %-wave dielectric may not be the entire solution as this background may become amplified with the same gain as the signal from the analyte.
Some biochips are made using 3-dimensional analyte domains rather than the planar domains addressed in prior art; such domains may easily have a height that is ½ or more wavelengths. When placed upon a ¼-wave dielectric atop a reflective layer, amplification exists, but it is more complex as different parts of the domain experience constructive or destructive interference, resulting in some cases in distinct ring patterns if the domain is circularly domed. On average though, substantial amplification may be anticipated. At the plane of attachment for the analyte domains, constructive interference will exist both for points in the domain near this attachment plane, as well as for spurious background arising from within this plane.
In U.S. Pat. No. 6,174,683, Hahn et al. discuss a novel method for developing biochips that result in domed spots comprising a reactive target biomolecule immobilized in a hydrogel matrix. Probes with attached fluorophores can enter the matrix and combine with the target, resulting in a 3-dimensional fluorophore source distribution. Experiments have shown that this source distribution can be several optical emission wavelengths thick. An example is published in the paper “Optimized Approach for Microassay Scanning,” D. Rachlin, Proc. SPIE, Vol 4623, p. 13–26 (June 2002) in which a pattern or concentric rings is demonstrated in the image of a fluorescent hydrogel spot that lies atop a reflective aluminum substrate. The rings are created by the alternating constructive and destructive interference over the varying thickness of the spot. The paper analyzes a ¼-wave approach and multi-wavelength thick substrate approaches.
Very generally, for 3-dimensional analyte domains, insofar as the total yield of amplified signal is concerned, there may be no “right” phase thickness of dielectric to promote amplification, so one may use a thickness of any phase with respect to a wavelength of interest.